Expression, purification, and spectral tuning of RhoGC, a retinylidene/guanylyl cyclase fusion protein and optogenetics tool from the aquatic fungus Blastocladiella emersonii

Abstract

RhoGC is a rhodopsin (Rho)-guanylyl cyclase (GC) gene fusion molecule that is central to zoospore phototaxis in the aquatic fungus It has generated considerable excitement because of its demonstrated potential as a tool for optogenetic manipulation of cell-signaling pathways involving cyclic nucleotides. However, a reliable method for expressing and purifying RhoGC is currently lacking. We present here an expression and purification system for isolation of the full-length RhoGC protein expressed in HEK293 cells in detergent solution. The protein exhibits robust light-dependent guanylyl cyclase activity, whereas a truncated form lacking the 17- to 20-kDa N-terminal domain is completely inactive under identical conditions. Moreover, we designed several RhoGC mutants to increase the utility of the protein for optogenetic studies. The first class we generated has altered absorption spectra designed for selective activation by different wavelengths of light. Two mutants were created with blue-shifted (E254D, λ = 390 nm; D380N, λ = 506 nm) and one with red-shifted (D380E, λ = 533 nm) absorption maxima relative to the wild-type protein (λ = 527 nm). We also engineered a double mutant, E497K/C566D, that changes the enzyme to a specific, light-stimulated adenylyl cyclase that catalyzes the formation of cAMP from ATP. We anticipate that this expression/purification system and these RhoGC mutants will facilitate mechanistic and structural exploration of this important enzyme.

Structure/function of the soluble guanylyl cyclase catalytic domain

“…In addition, the presence of the coiled-coil domain decreased overall activity (21 fold) in the presence of Mn 2+ , but increased overall activity (11 fold) in the presence of Mg 2+ . This is in agreement with earlier studies [65] suggesting that other domains of RhoGC modulate the activity of the cyclase domain, and has also been proposed for human GC-1 [42,43,63]. The structure of monomeric GC Rho (Fig.…”

“…The homodimeric transmembrane protein senses light through a rhodopsin domain (residues 176-388) and transduces the signal via a coiled-coil domain to the cytosolic catalytic GC Rho domain (residues 443-626) where GTP can be cyclized for phototaxis [64,65]. Kumar et al expressed and purified two constructs containing the catalytic domain: GC Rho and GCwCC Rho (with coiled-coil domain), which are monomeric in solution and surprisingly both catalytically active.…”

Purification and Characterization of RhoPDE, a Retinylidene/Phosphodiesterase Fusion Protein and Potential Optogenetic Tool from the Choanoflagellate Salpingoeca rosetta

“…These data are similar to those obtained for the related fusion protein RhoGC from B. emersonii , which is also predicted to have eight transmembrane helices by hydropahy analysis and has been shown experimentally to have an even number of transmembrane segments with N- and C-termini on the cytoplasmic surface of the plasma membrane. 6,15 Thus, both fusion proteins share this structural feature.…”

“…The yield of protein from the 1D4 column was increased significantly by utilizing a E8′A mutant 1D4 epitope tag, which lowers the protein’s affinity for the antibody enough that it can be eluted efficiently from the resin with the wild-type peptide. Tandem purification with both antibodies, which we have used previously 15 to purify a rhodopsin/guanylyl cyclase fusion protein (RhoGC) found in the aquatic fungus B. emersonii , ensures that the full-length protein is purified.…”

“…14,15 The protein was reconstituted with the retinal chromophore by incubating the cell suspension with 10 μ M ATR for 30 min in the dark at 4 °C. All procedures following the addition of retinal were performed in the dark under dim red lights.…”